1. Field of the Invention
This invention relates in general to devices and methods for determination of substances and more particularly to methods and devices for assays of binding or bindable substances. Still further, this invention relates to methods and devices for quantitative assays of such binding or bindable substances.
2. Description of the Prior Art
In the past many tests have been provided for determination of binding and bindable substances of various types. Examples of such binding and bindable substances and the substances bound thereto or thereby are antibodies-antigens and other binding proteins-protein bindable substances.
An example of a particular type of demonstration or detection process (assay) used in the past is described in U.S. Pat. No. 3,654,090 to Schuurs et al. This patent describes a test for determination of either an antigen or an antibody. In one example of the 3,654,090 patent, a quantity of antigen is attached to an enzyme and a quantity of antibody is insolubilized by attachment to an insoluble carrier. The enzyme-labelled antigen and the insolubilized antibody are mixed with an unlabeled antigen (the assay substance). By controlling the amount of insolubilized antibody and enzyme-labeled antigen, some or all of the enzyme-labelled antigen is not attached to antibody when unlabeled antigen is present. Some or none of the enzyme-labelled antigen is attached. After mixing, the insoluble material is separated from the soluble material which includes any unattached enzyme-labelled antigen. Centrifuging or washing the insoluble material achieves this separation. An enzyme-reactive agent is then added to either the insoluble or soluble portions to assay the enzyme activity. In this manner, the presence of unlabeled antigen (the assay substance) is determined.
U.S. Pat. No. 3,791,932 to Schuurs et al. describes a similar process for assaying either a binding protein or a substance which is specifically bound by such a protein. In this assay method one of these components is insolubilized and then mixed with the component to be determined, binding the protein to its complement. Next, the solid phase of the insoluble form is separated and reacted with a coupling product of the substance to be determined with an enzyme; i.e., an enzyme-labelled component is mixed with the solid phase such that the enzyme-labeled component will also be bound. The presence of the component to be determined in the first mixing step will prevent coupling of the coupling product (enzyme-component) with at least some of the insolubilized binding components. Determination of the enzyme activity of either the solid or liquid phase resulting from the last mixture will determine the presence of the described components. U.S. Reissue Pat. No. 29,169 is related to U.S. Pat. No. 3,791,932.
Other patents showing similar processes for the determination of binding or bindable substances are shown in U.S. Pat. Nos. 3,839,153 to Schuurs, et al.; 3,850,752 to Schuurs, et al; and 4,016,043 to Schuurs, et al. Each of these patents are similar in that one component is labeled with an enzyme and the other is made insoluble. After mixing the labelled component, the insoluble component and an unlabeled, to be determined, component, the soluble component is separated and the enzyme activity of either the soluble or insoluble portions determines the presence of the unlabeled component.
As described in U.S. Pat. No. 3,966,897 to Renn et al. the same types of competitive and non-competitive bio-assay processes have been performed by radioactively labeling the component instead of enzyme labeling the component. In this type of assay radioactivity is measured instead of enzyme activity. As also described in the 3,966,879 patent, indicator dyes can be attached to the components and then directly measured by visual examination, fluorimetry, spectrophotometry, refractometry or the like. Many of these tests utilize refractive index or radiation intensity or quantitatively measure the assay.
All of these assay procedures involve the reaction of a test substance with an appropriate reagent, for example, hormone-antibody, antigen-antibody, enzyme-substrate, or their converse reactions, together with direct or indirect quantitative measurement of the amount of reagent so reacted by the measurement of a characteristic of a reagent (or of another substance, an indicator, which has reacted with the reagent) such as color, radioactivity, or other physical characteristics.
As shown in U.S. Pat. No. 3,966,897 to Renn et al., in some instances the reagent is immobilized or fixed to a medium into which test substances can be diffused. In the Renn et al. patent, test substance is diffused into a gel medium in which reagent is immobilized against diffusion. One way of immobilizing the reagents in the gel is by absorbing the reagent onto talc, porous glass beads, hydroxyapatite, zirconyl phosphate, charcoal polyethylene, polypropylene, polystyrene particles or the like. After binding the reagent to such a material a conventional assay using a labeled material is obtained by first diffusing test substance into the reagent zone and then using indicators for reacting with the remaining unattached reagent. After excess indicator has been removed, determination of the attached indicator completes the assay.
U.S. Pat. Nos. 3,527,712 to Renn et al. and 3,975,162 to Renn describe processes for applying reagents to mediums such as described in the 3,966,897 patent and preparing gels for use in such mediums.
U.S. Pat. No. 3,654,084 to Moelker describes a process for preparing stable, water-soluble enzyne conjugates.
In the past, avidin and biotin have been coupled with various antigens and antibodies for the purpose of further coupling these antigens and antibodies to other components which could not otherwise be coupled thereto. This has produced antigen or antibody coupling products specific for two or more normally unrelated components.
A particular problem with the assay methods and apparatus which have been used in the past is that they require washing, centrifuging or other separation techniques for separating an insolubilized component from a labeled component. This requires extensive time and labor. Also, equipment for automatically centrifuging and washing a large number of test pieces is expensive. Other test equipment utilized in these past assay methods has been complex and expensive as well.
Another problem with assay methods and devices used in the past is that they are not portable. Thus, field testing has not been easily accomplished. Additionally, most devices for assays have not been simple to assemble or use. Only experts can, therefore, perform most of these assays. Furthermore, most assay chemicals and devices do not have a long shelf life and require expert mixing or preparation.
Still another problem with assay methods and devices used in the past is that they have not been quantitatively accurate, or if quantitatively accurate, the procedure has been complex and requires extensive detection equipment. This is because most quantitative assays are performed by measuring radiation intensity or refractive index.